Anti-Viral Therapy, Cutting Edge MarinBio Science Newsletter, Targeted Protein Degradation

Researchers Unveil New Insights into Trivalent PROTACs and the Hook Effect

Molecular illustration of a Trivalent PROTAC linking von Hippel–Lindau (VHL) and Bromodomain-containing 2 (BRD2) proteins.

Researchers from AstraZeneca have unveiled new insights into the behavior of trivalent proteolysis-targeting chimeras (PROTACs), challenging previous assumptions about their efficacy in targeted protein degradation. A study by Imaide et al. (2021), published in Nature Chemical Biology, explored how increasing PROTAC valency might influence the formation of ternary complexes between PROTAC, the protein of interest (POI), and E3 ligase. The researchers introduced SIM1, a trivalent von Hippel–Lindau (VHL)-based PROTAC, which demonstrated superior degradation of BRD2 compared to its bivalent predecessor, MZ1. However, subsequent investigations by AstraZeneca scientists have challenged these assumptions about the efficacy of trivalent PROTACs. Their study unveiled new insights into the behavior of SIM1, suggesting that the occurrence of the hook effect is influenced by various factors beyond PROTAC valency, including cellular context and protein modifications. This new data significantly impacts our understanding of PROTAC efficacy and behavior. The research, which focused on the degradation of bromodomain and extra-terminal domain (BET) proteins, revealed that the presence or absence of a hook effect—a phenomenon where drug efficacy decreases at higher concentrations—is influenced by multiple factors beyond PROTAC valency alone. The researchers compared SIM1 and MZ1 degradation profiles in various cell lines, including naïve and CRISPR/Cas9-engineered HEK293 cells expressing HiBiT-tagged BRD2 and BRD4. They used Western blotting, Nano-Glo HiBiT lytic detection assays, and immunostaining to assess protein degradation and localization.

Key Findings

SIM1 exhibited different degradation preferences for BRD2 and BRD4 across cell lines.
Naïve HEK293 cells showed a pronounced hook effect for BRD2 and a lesser effect for BRD4, contradicting previous reports.
PROTAC efficacy varied significantly between low efflux (MV4-11) and high efflux (HEL92.1.7) cell lines, likely due to differences in MDR1 expression.
The hook effect onset occurred at different concentrations for BRD2 and BRD4, independent of VHL ligase levels.

The study concludes that optimizing PROTAC design requires considering these variables to maximize therapeutic potential. This research contributes to a more nuanced understanding of PROTAC behavior and efficacy, potentially leading to improved therapeutic strategies in targeted protein degradation.